The present invention, in some embodiments thereof, relates to adhesive biopolymers and uses thereof.
The replacement or repair of damaged or diseased tissues or organs by implantation has been, and continues to be, a long-standing goal of medicine towards which tremendous progress has been made. Working toward that goal, there is an increasing interest in tissue engineering techniques where biocompatible, biodegradable materials are used as adhesives.
A variety of adhesives found in nature, such as barnacle glue, appear to have excellent polymerization and mechanical properties. However, development of natural product based glues has been hampered by the ability to purify appreciable quantities of such materials, as well as persistent concerns about the triggering of an immune response by foreign glycoproteins.
Owing to the above-described limitations, considerable development effort has been directed towards finding a suitable synthetic composition operative as a tissue glue. To this end, cyanoacrylates, polyurethanes, polymethylmethacrylates, among other synthetic polymers, have been investigated as tissue glues. Each of these synthetic compositions has met with limited success owing to a variety of problems such as toxic degradation products, poor mechanical properties, cure exotherms that overheat surrounding tissue, and not being biodegradable. The replacement or repair of damaged or diseased tissues or organs by implantation has been, and continues to be, a long-standing goal of medicine towards which tremendous progress has been made. Working toward that goal, there is an increasing interest in tissue engineering techniques where biocompatible, biodegradable materials are used as adhesives.
Many elastomeric proteins are found in a diverse range of animal species and tissues and possess rubber-like elasticity, undergoing high deformation without rupture, storing the energy involved in deformation, and then returning to their original state when the stress is removed [33]. Protein elasticity remains to be fully described due to the large size and complexity of these proteins which has led to difficulties in isolation and purification [32]. Only a few elastomeric proteins, especially elastin, abductin, resilin and some spider silks, have been studied for mechanical and biochemical properties, and their potential as biomaterials for industrial and biomedical applications has been documented [16, 18, 34, 35]. In nature, resilin and elastin have achieved near-perfect elasticity, and these two elastomeric proteins can be stretched more than twice their original length and recover more than 90% of the deformation energy once the stretching (compression) force is removed [32]. The latter property is called resilience, hence the name “resilin”.
Resilin is found in specialized cuticle regions in many insects, especially in areas where high resilience and low stiffness are required, or as an energy storage system. It is best known for its roles in insect flight and the remarkable jumping ability of fleas and spittlebugs. The protein was initially identified in 1960 by Weis-Fogh who isolated it from cuticles of locusts and dragonflies and described it as a rubber-like material.
Resilin displays unique mechanical properties that combine reversible deformation with very high resilience. It has been reported to be the most highly efficient elastic material known.
Ardell et al. identified the gene product CG15920 as a tentative D. melanogaster resilin precursor, which had the three exons: N-terminal region (exon 1, containing signal peptide and elastic repeats), chitin-binding domain (ChBD; exon 2), and C-terminal region (exon 3, with other types of elastic repeats) [9]. Exon 2 in D. melanogaster resilin, identified as ChBD type R&R-2, has been studied experimentally for chitin binding to resilin [12]. Exon 1 and exon 3 domains contain regions of highly repetitive segments (elastic repeats), which are rich in proline and glycine similar to most proteins that have long-range elasticity.
Based on the first exon of CG15920, a recombinant resilin-like protein (rec1) was successfully expressed, purified and photo-chemically cross-linked to form a resilient elastic biomaterial [16]. Surface-induced assembly of this biomaterial has been investigated through direct imaging using AFM [17], and the recombinant materials from exon 1 exhibited potentially useful mechanical and cell adhesion behavior [18].
The full length resilin containing all three exons in D. melanogaster CG15920 have also expressed, purified without affinity tags, and cross-linked by HRP (horseradish peroxidase). A high degree of disorder and high resilience were exhibited by the full length resilin (exon 1+exon 2+exon 3) [12].
Resilin and composites thereof have been described in International Application No. WO 2009/069123.
Elastic proteins often contain repeat sequences forming elastomeric domains, and additional domains that form intermolecular cross-links [39, 40]. Elasticity depends on the length of elastic sequence and the extent of cross-linking. The presence of a network of cross-links (covalent or noncovalent) is a common feature to most of these proteins [14, 37].
Natural resilin is cross-linked in insect cuticle via di-tyrosine formation via enzymes, resulting in an almost perfect 3D elastomer. Both enzyme-based and Ru-based methods have been reported for resilin polymerization [12, 16].
Cross-linking of resilin proteins has been reported to produce high molecular weight cross-linked polymeric material by using horseradish peroxidase, an enzyme known to catalyze di-tyrosine formation, and present in extracts of resilin from the adult desert locust (Schistocerca gregaria) [4, 41].